Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis

Mitochondria are particularly vulnerable to oxidative stress, and mitochondrial swelling and vacuolization are among the earliest pathologic features found in two strains of transgenic amyotrophic lateral sclerosis (ALS) mice with SOD1 mutations. Mice with the G93A human SOD1 mutation have altered electron transport enzymes, and expression of the mutant enzyme in vitro results in a loss of mitochondrial membrane potential and elevated cytosolic calcium concentration. Mitochondrial dysfunction may lead to ATP depletion, which may contribute to cell death. If this is true, then buffering intracellular energy levels could exert neuroprotective effects. Creatine kinase and its substrates creatine and phosphocreatine constitute an intricate cellular energy buffering and transport system connecting sites of energy production (mitochondria) with sites of energy consumption, and creatine administration stabilizes the mitochondrial creatine kinase and inhibits opening of the mitochondrial transition pore. We found that oral administration of creatine produced a dose-dependent improvement in motor performance and extended survival in G93A transgenic mice, and it protected mice from loss of both motor neurons and substantia nigra neurons at 120 days of age. Creatine administration protected G93A transgenic mice from increases in biochemical indices of oxidative damage. Therefore, creatine administration may be a new therapeutic strategy for ALS.     

The creatine kinase system and pleiotropic effects of creatine

The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure–function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans.     

Neuroprotective effects of naturally occurring biflavonoids

We examined neuroprotective effects of naturally occurring biflavonoids on oxidative stress-induced and amyloid beta peptide-induced cell death in neuronal cells. Among the nine biflavonoids tested, amentoflavone, ginkgetin, and isoginkgetin exhibited strong neuroprotection against cytotoxic insults induced by oxidative stress and amyloid beta, suggesting their therapeutic potential against neurodegenerative diseases, including ischemic stroke and Alzheimer's disease.     

Neuroprotective effects of blockers for T-type calcium channels

Background

Activation of the liver × receptors (LXRs) by exogenous ligands stimulates the degradation of β-amyloid 1–42 (Aβ42), a peptide that plays a central role in the pathogenesis of Alzheimer's disease (AD). The oxidized cholesterol products (oxysterols), 24-hydroxycholesterol (24-OHC) and 27-hydroxycholesterol (27-OHC), are endogenous activators of LXRs. However, the mechanisms by which these oxysterols may modulate Aβ42 levels are not well known.

Results

We determined the effect of 24-OHC and/or 27-OHC on Aβ generation in SH-SY5Y cells. We found that while 27-OHC increases levels of Aβ42, 24-OHC did not affect levels of this peptide. Increased Aβ42 levels with 27-OHC are associated with increased levels of β-amyloid precursor protein (APP) as well as β-secretase (BACE1), the enzyme that cleaves APP to yield Aβ. Unchanged Aβ42 levels with 24-OHC are associated with increased levels of sAPPα, suggesting that 24-OHC favors the processing of APP to the non-amyloidogenic pathway. Interestingly, 24-OHC, but not 27-OHC, increases levels of the ATP-binding cassette transporters, ABCA1 and ABCG1, which regulate cholesterol transport within and between cells.

Conclusion

These results suggest that cholesterol metabolites are linked to Aβ42 production. 24-OHC may favor the non-amyloidogenic pathway and 27-OHC may enhance production of Aβ42 by upregulating APP and BACE1. Regulation of 24-OHC: 27-OHC ratio could be an important strategy in controlling Aβ42 levels in AD.     

Neuroprotective effects of quercetin in diabetic rat retina

Diabetic retinopathy (DR) is a severe complication of diabetes and the leading cause of blindness among working adults worldwide. DR is being widely recognized as a neurodegenerative disease of the retina, since, retinal neurons are damaged soon after diabetes onset. Diabetes-induced oxidative stress is considered as central factor that dysregulates neurotrophic factors and activates apoptosis, thereby damages neurons in the diabetic retina. Flavonoids being a powerful antioxidant have been considered to protect neurons in diabetic retina. The purpose of this study was to analyze the beneficial effects of flavonoid, quercetin to protect neurons in the diabetic rat retina. We quantitated the expression levels of BDNF, NGF, TrkB, synaptophysin, Akt, Bcl-2, cytochrome c and caspase-3 using Western blotting techniques in the diabetic retina with and without quercetin treatments and compared with non-diabetic rats. In addition, we employed ELISA techniques to determine the level of BDNF. Caspase-3 activity and the level of glutathione were analyzed by biochemical methods. Our results indicate that quercetin treatment to diabetic rats caused a significant increase in the level of neurotrophic factors and inhibited the level of cytochrome c and caspase-3 activity in the diabetic retina. Furthermore, the level of an anti-apoptotic protein Bcl-2 was augmented in quercetin treated diabetic retina. Thus, quercetin, may protect the neuronal damage in diabetic retina by ameliorating the levels of neurotrophic factors and also by inhibiting the apoptosis of neurons. Therefore, this study suggests that quercetin can be a suitable therapeutic agent to prevent neurodegeneration in diabetic retinopathy.     

Cerebral energetic effects of creatine supplementation in humans

There has been considerable interest in the use of creatine (Cr) supplementation to treat neurological disorders. However, in contrast to muscle physiology, there are relatively few studies of creatine supplementation in the brain. In this report, we use high-field MR (31)P and (1)H spectroscopic imaging of human brain with a 7-day protocol of oral Cr supplementation to examine its effects on cerebral energetics (phosphocreatine, PCr; ATP) and mitochondrial metabolism (N-acetyl aspartate, NAA; and Cr). We find an increased ratio of PCr/ATP (day 0, 0.80 +/- 0.10; day 7, 0.85 +/- 09), with this change largely due to decreased ATP, from 2.7 +/- 0.3 mM to 2.5 +/- 0.3 mM. The ratio of NAA/Cr also decreased (day 0, 1.32 +/- 0.17; day 7 1.18 +/- 0.13), primarily from increased Cr (9.6 +/- 1.9 to 10.1 +/- 2.0 mM). The Cr-induced changes significantly correlated with the basal state, with the fractional increase in PCr/ATP negatively correlating with the basal PCr/ATP value (R = -0.74, P < 0.001). As NAA is a measure of mitochondrial function, there was also a significant negative correlation between basal NAA concentrations with the fractional change in PCr and ATP. Thus healthy human brain energetics is malleable and shifts with 7 days of Cr supplementation, with the regions of initially low PCr showing the largest increments in PCr. Overall, Cr supplementation appears to improve high-energy phosphate turnover in healthy brain and can result in either a decrease or an increase in high-energy phosphate concentrations.     

利格列汀对小鼠脑缺血/再灌注损伤的神经保护作用*

目的: 评估二肽基肽酶4(DPP-4)抑制剂利格列汀对小鼠脑缺血/再灌注(I/R)损伤的神经保护作用。方法: BALB/c小鼠随机分为Sham组、I/R组和利格列汀(2.5、5和10 mg/kg) +I/R组,每组均为8只小鼠。不同剂量利格列汀组小鼠均在I/R前3周连续灌胃给药。采用小鼠脑中动脉闭塞(MCAO)1 h诱导I/R损伤模型,再灌注24 h评估神经功能缺损(n=8)和及梗死体积(n=4);再灌注48 h处死小鼠,检测脑组织中谷胱甘肽(GSH)、丙二醛(MDA)、磷酸化肌醇3激酶(PI3K)、磷酸化蛋白激酶 B(p-Akt)和雷帕霉素靶蛋白(mTOR)含量(n=4)。结果: 与I/R组相比,利他列汀预处理组小鼠再灌注24 h后,神经功能缺损评分和梗死体积明显降低(P＜0.05);小鼠再灌注48 h后,脑内MDA含量明显降低(P＜0.05),而GSH、PI3K、p-Akt和mTOR水平明显升高(P＜0.05)。结论: 利格列汀对I/R小鼠具有神经保护作用,可能是通过激活PI3K/AKT/mTOR通路发挥的作用。     

Neuroprotective effects of hydroxyethylpuerarin against focal cerebral ischemia-reperfusion in rats

Our present study was performed to investigate whether hydroxyethylpuerarin (HEP) has a neuroprotective effect on brain injury after focal cerebral ischemia/reperfusion by middle cerebral artery occlusion (MCAO) in adult male Wistar rats. Animals were subjected to one hour of middle cerebral artery occlusion and 48 hours of reperfusion with the pretreatment of drugs (HEP 15, 30, 60 mg/ kg or nimodipine 0.4 mg/kg i.v.) or vehicle. The behavioral tests were used to evaluate the damage to central nervous system. The percentage of brain infarct area was assessed in the brain slices stained with 2% solution of 2, 3, 5-triphenyl tetrazolium chloride (TTC). The pathologic histological changes were observed by H&E staining and the occurrence of apoptosis was determined by flow cytometry. The results showed that pretreatment with HEP at doses of 15, 30, 60 mg/kg exhibited significant neuroprotective effects on rats against focal cerebral ischemia-reperfusion injury by markedly decreasing neurological deficit scores and the percentage of infarct area, reducing necrosis and apoptosis of neurons. All these findings suggest that HEP might provide neuroprotection against focal cerebral ischemia/reperfusion injury probably through its antioxidant and anti-inflammatory property.     

Neuroprotective effects of interleukin-6 on NMDA-induced rat retinal damage

This study shows that interleukin-6 (IL-6) combined with soluble interleukin-6 receptors (sIL-6R) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Eyes pretreated with a combined injection of IL-6 and sIL-6R had NMDA administered into the vitreous cavity. Morphometric analysis and retrograde labeling analysis found that pretreatment with either IL-6 or sIL-6R alone did not bring about any neuroprotective effect. However, pretreatment with a combined administration of IL-6 and sIL-6R induced a significant neuroprotective effect against NMDA-induced retinal damage. Apoptotic changes in the retina were assessed by the TUNEL method. The results indicated that pretreatment with IL-6 combined with sIL-6R prevents NMDA-induced apoptosis. Western blotting studies demonstrated upregulation of gp130 expression in the NMDA-injected retina. Present studies suggest that IL-6 combined with sIL-6R provides a neuroprotective effect on NMDA-induced retinal damage.     

Neuroprotective effects of amiodarone in a mouse model of ischemic stroke

Background

Ion channels play a crucial role in the development of ischemic brain injury. Recent studies have reported that the blockade of various types of ion channels improves outcomes in experimental stroke models. Amiodarone, one of the most effective drugs for life-threatening arrhythmia, works as a multiple channel blocker and its characteristics cover all four Vaughan-Williams classes. Although it is known that amiodarone indirectly contributes to preventing ischemic stroke by maintaining sinus rhythm in patients with atrial fibrillation, the direct neuroprotective effect of amiodarone has not been clarified. The purpose of this study was to investigate the direct effect of amiodarone on ischemic stroke in mice.

Methods

Focal cerebral ischemia was induced via distal permanent middle cerebral artery occlusion (MCAO) in adult male mice. The amiodarone pre-treatment group received 50 mg/kg of amiodarone 1 h before MCAO; the amiodarone post-treatment groups received 50 mg/kg of amiodarone immediately after MCAO; the control group received vehicle only. In addition, the sodium channel opener veratrine and selective beta-adrenergic agonist isoprotelenol were used to elucidate the targeted pathway. Heart rate and blood pressure were monitored perioperatively. Infarct volume analysis was conducted 48 h after MCAO. The body asymmetry test and the corner test were used for neurological evaluation.

Results

Amiodarone pre-treatment and post-treatment reduced the heart rate but did not affect the blood pressure. No mice showed arrhythmia. Compared with the control group, the amiodarone pre-treatment group had smaller infarct volumes (8.9?±?2.1% hemisphere [mean?±?SD] vs. 11.2?±?1.4%; P?<?0.05) and improved functional outcomes: lower asymmetric body swing rates (52?±?17% vs. 65?±?18%; P?<?0.05) and fewer left turns (7.1?±?1.2 vs. 8.3?±?1.2; P?<?0.05). In contrast, amiodarone post-treatment did not improve the outcomes after MCAO. The neuroprotective effect of amiodarone pre-treatment was abolished by co-administration of veratrine but not by isoproterenol.

Conclusions

Amiodarone pre-treatment attenuated ischemic brain injury and improved functional outcomes without affecting heart rhythm and blood pressure. The present results showed that amiodarone pre-treatment has neuroprotective effects, at least in part, via blocking the sodium channels.

     

Neuroprotective effects of semax in MPTP-induced disturbances of brain dopamine system

Effects of an ACTH (4-10) analogue Semax (MEHFPGP) on behaviour of white rats with MPTP-induced disturbances of brain DA-system have been studied. It was shown that MPTP administration (25 mg/kg) reduced motor activity and auhmented the anxiety level in rats. Semax administration (daily intranasal 0.2 mg/kg) attenuated behaviour disturbances induced by neurotoxin. The observed protective action of Semax in rats with MFTP-induced DA system disturbances may be due to both its modulating influence on the brain DA system and peptide neuroprotective effects.     

Neuroprotective effects of atorvastatin against glutamate-induced excitotoxicity in primary cortical neurones

Statins [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors] exert cholesterol-independent pleiotropic effects that include anti-thrombotic, anti-inflammatory, and anti-oxidative properties. Here, we examined direct protective effects of atorvastatin on neurones in different cell damage models in vitro. Primary cortical neurones were pre-treated with atorvastatin and then exposed to (i) glutamate, (ii) oxygen-glucose deprivation or (iii) several apoptosis-inducing compounds. Atorvastatin significantly protected from glutamate-induced excitotoxicity as evidenced by propidium iodide staining, nuclear morphology, release of lactate dehydrogenase, and mitochondrial tetrazolium metabolism, but not from oxygen-glucose deprivation or apoptotic cell death. This anti-excitototoxic effect was evident with 2-4 days pre-treatment but not with daily administration or shorter-term pre-treatment. The protective properties occurred independently of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition because co-treatment with mevalonate or other isoprenoids did not reverse or attenuate neuroprotection. Atorvastatin attenuated the glutamate-induced increase of intracellular calcium, which was associated with a modulation of NMDA receptor function. Taken together, atorvastatin exerts specific anti-excitotoxic effects independent of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition, which has potential therapeutic implications.     

Neuroprotective effects of erythropoietin on 6-hydroxydopamine-treated ventral mesencephalic dopamine-rich cultures

Parkinson's disease (PD) is a neurodegenerative disorder with motor symptoms caused by the loss of dopaminergic (DA) cells and consequently dopamine release in the nigrostriatal system. In vivo and in vitro 6-hydroxydopamine (6-OHDA) PD models are widely used to study the effect of striatal dopamine depletion as well as novel neuroprotective or restorative therapeutic strategies for PD. In the present study, we investigated in vitro the toxicity of 6-OHDA on DA neurons derived from E14 rat ventral mesencephalon (VM) and the neuroprotective efficiency of erythropoietin (Epo) on VM-derived cell cultures against 6-OHDA toxicity. Using E14 VM-derived DA-rich primary cultures, we could demonstrate that 6-OHDA toxicity works in a time-and concentration-dependent way, and leads to cell death not only in DA cells but also in non-DA cells in direct relation to concentration and incubation times. In addition, we found that 6-OHDA toxicity induces caspase-3 activation and an increment of intracellular reactive oxygen species (ROS) in VM-derived cultures. When 6-OHDA-treated VMs were cultured in the presence of the anti-apoptotic protein erythropoietin (Epo), the total neuronal population, including the DA neurons, was protected. However, untreated VM cultures exposed to Epo showed an increase in the total neuronal population, but not an additional increase in DA neuron cell number.These findings suggest that 6-OHDA toxicity is time and concentration-dependent and does not exclusively affect DA neurons. In high concentration and long incubation times, 6-OHDA influences the survival of other neuronal and non-neuronal cell populations derived from the VM cultures. 6-OHDA toxicity induces caspase-3 activation, indicating cell death via the apoptotic pathway which could be restricted or even prevented by pre-exposure to Epo, known to interact via the apoptotic pathway. Our results support and expand on previous findings showing that Epo is an interesting candidate molecule to mediate neuroprotective effects on DA neurons in PD. Furthermore, it could be used in promoting the survival of DA neurons after transplantation in clinical trials.     

Neuroprotective effects of valproic acid following transient global ischemia in rats

AimsA growing number of studies demonstrate that valproic acid (VPA), an anti-convulsant and mood-stabilizing drug, is neuroprotective against various insults. This study investigated whether treatment of ischemic stroke with VPA ameliorated hippocampal cell death and cognitive deficits. Possible mechanisms of action were also investigated.Main methodsGlobal cerebral ischemia was induced to mimic ischemia/reperfusion (I/R) damage. The pyramidal cells within the CA1 field were stained with cresyl violet. Cognitive ability was measured 7 days after I/R using a Morris water maze. The anti-inflammatory effects of VPA on microglia were also investigated by immunohistochemistry. Pro-inflammatory cytokine production was determined using enzyme-linked immunosorbent assays (ELISA). Western blot analysis was performed to determine the levels of acetylated H3, H4 and heat shock protein 70 (HSP70) in extracts from the ischemic hippocampus.Key findingsVPA significantly increased the density of neurons that survived in the CA1 region of the hippocampus on the 7th day after transient global ischemia. VPA ameliorated severe deficiencies in spatial cognitive performance induced by transient global ischemia. Post-insult treatment with VPA also dramatically suppressed the activation of microglia but not astrocytes, reduced the number of microglia, and inhibited other inflammatory markers in the ischemic brain. VPA treatment resulted in a significant increase in levels of acetylated histones H3 and H4 as well as HSP70 in the hippocampus.SignificanceOur results indicated that VPA protected against hippocampal cell loss and cognitive deficits. Treatment with VPA following cerebral ischemia probably involves multiple mechanisms of action, including inhibition of ischemia-induced cerebral inflammation, inhibition of histone deacetylase (HDAC) and induction of HSP.     

学习训练对谷氨酸单钠所致大鼠海马损伤的神经保护作用

目的：探讨学习训练对谷氨酸神经毒性的保护作用。方法：在SD大鼠生后第3～9d腹腔注射谷氨酸单钠复制谷氨酸毒性模型,在1月龄和2月龄时训练大鼠学会以明暗辨别来获得食物,3月龄时取脑,在光镜下计数海马内存活神经元数,电镜下观察海马CA1区的超微结构,并计数突触数,测量突触活性带长度。结果：学习训练组海马CA3区和CA4区内的存活神经元数、海马CA1区内的突触数和突触活性带长度均大于非学习组,结论：结果提示学习训练可在一定程度上减轻MSG对海马的损伤。